Photon-counting X-ray and Optical Tomography for Preclinical Cancer Research ABSTRACT Preclinical imaging is a critical tool in cancer research. Since cancer exhibits very complex spatiotemporal features, there is a strong need for the development of novel imaging technologies to characterize cancerous tissues and their microenvironments. For this purpose, multimodal imaging has the best potential to provide anatomical, functional and molecular information concurrently in live and intact animals. Of our primary interest, human epidermal growth factor receptor 2 (HER2) expression has prognostic and predictive values in breast cancer. Currently, therapeutic monoclonal anti-HER2 antibodies that inhibit receptor dimerization are FDA- approved. However, an increasingly more complex view of the role of HER2 in breast cancer has emerged from genome sequencing that highlights the importance of inter- and intra-tumor heterogeneity in therapy resistance. Thus, there is a clear need for a non-invasive preclinical imaging modality that is capable of monitoring the interplay between HER2 receptor expression level, targeted drug delivery, and tumor response. The overall goal of this project is to develop a hybrid x-ray and optical prototype for High-dimensional Optical Tomography (HOT) Guided-by Energy-resolved Micro-CT (GEM), visualize and quantitate breast tumor heterogeneity, HER2 expression and dimerization, and therapeutic response in preclinical models. On the x- ray side, photon-counting micro-CT records individual x-ray photons and their energy levels, and enables chemically-specific material decomposition. As a result, a mouse anatomy can be represented in terms of water, lipid, bone, Calcium, Iodine, and Gadolinium. On the optical side, optical molecular tomography maps the distribution of functional biomarkers and molecular probes. Of great importance to targeted therapy, with in vivo Macroscopy Fluorescence Lifetime Frster Resonance Energy Transfer (MFLI-FRET) imaging, our recent results demonstrate that quantitative MFLI-FRET signals correlate strongly with intracellular drug delivery at the pathological site as validated via ex vivo immunohistochemistry analysis. Synergistically, basis materials resolved with photon-counting micro-CT can be related to unique optical properties, and used to correct a heterogeneous optical background for quantitative optical molecular tomography. Furthermore, contrast- enhanced micro-CT can identify regions of interest to regularize optical molecular tomography. The specific aims are to (1) prototype a hybrid HOTGEM system for comprehensive and synergistic x-ray and optical imaging, (2) develop joint methods for image reconstruction from datasets in multi-contrasts collected with the HOTGEM system, and (3) characterize breast cancer in xenograft systems with varying levels of HER2 and HER2-activating mutations using the HOTGEM system. Upon completion, the proposed HOTGEM system will have been validated to offer 50m x-ray resolution for material decomposition and 100m optical resolution for target localization in co-registration within 30 minutes for each hybrid in vivo scan, demonstrated to be a breakthrough for tomographic HER2 imaging, and ready for technology transfer and commercial translation.